How the "base editor" works
"Base Editor" based on CRISPR/Cas9 prevented anemia in human embryos
Daria Spasskaya, N+1
Chinese researchers have successfully corrected a mutation in the beta-globin gene of human embryos that leads to the development of anemia. As a tool for genome editing, scientists used a "base editor" developed on the basis of the CRISPR/Cas9 system. With its help, it was possible to purposefully introduce a single-nucleotide replacement into the genome in a quarter of cases. The study was published in the journal Protein & Cell, and it is also described in an editorial in the journal Nature.
Mutations in the genes encoding hemoglobin chains are the cause of the development of thalassemia – anemia caused by a violation of hemoglobin synthesis. Single-nucleotide substitution of adenine for guanine in the regulatory region of the HBB gene (HBB -28 A>G) encoding beta-globin is one of the three most common causes of beta-thalassemia in China and Southeast Asia. This mutation leads to a decrease in the expression of the HBB gene and, as a consequence, to hemoglobin deficiency in its carrier.
Chinese researchers from Sun Yat-sen University managed to correct this mutation in human embryos by turning G back to A using a new CRISPR/Cas9-based genome editing tool. The editing efficiency was 40 percent at the embryo level and 23 percent at the individual cell level.
The new system, called the "base editor" (a publication on the development of this method can be found here), uses a catalytically inactive dCas9 protein that is unable to cut DNA. Such a protein in combination with a guide RNA serves as a means of delivering the "working" domain sewn to it to a certain sequence in DNA.
The working part in the "editor" is the enzyme cytosine deaminase, which "detaches" the amino group from cytosine, leaving uracil in its place. As a result, an "incorrect" U:G pair is formed in DNA (uracil opposite guanine), which is recognized by the repair system of unpaired bases. An additional modification of the method made it possible to achieve that it is G. that is repaired in the chain. This leads to the formation of a "correct" pair U:A, which after a round of replication turns into T:A. The scheme of the method is presented below, and you can refresh the principle of operation of the CRISPR/Cas9 system here.
The principle of operation of the "base editor". Inactive Cas9 (dCas9) is marked in blue, cytosine deaminase is red, and guide RNA is green. After binding the hybrid protein to the desired DNA site, cytosine deaminase converts cytosine into uracil, as a result of which, after repair and a round of replication, the G:C pair is replaced by A:T
A little earlier, another research group demonstrated that this method is applicable in trypronuclear zygotes ("defective" embryos obtained by artificial insemination containing a triple set of chromosomes). In the new work, the researchers needed embryos containing the HBB -28 (A>G) mutation in two copies. Such embryos were obtained by cloning – fusion of a somatic cell of a patient with thalassemia with a nuclear-free egg.
Shortly after the merger, the researchers injected the mRNA of the "editor" and the guide RNA for it into the resulting unicellular embryos. 48 hours later, after the developing embryos had managed to go through three or four divisions, the authors analyzed whether the editing had passed. The analysis was carried out by sequencing a site with a mutation that needed to be corrected, and ten additional sites where the "editor" could potentially be involved.
In the first experiment, the analysis of the total DNA of 22 embryos showed that in 9 cases the editing was correct, and G was replaced by A. In another , G was replaced by C. Thus, successful editing took place in 40 percent of the embryos.
In the second experiment, the efficiency of editing was evaluated at the level of individual embryo cells. In this case, the success rate was 11 out of 48, while in eight cases the editing took place along both chains, that is, both copies of the HBB gene were corrected. However, such a discrepancy between the result at the level of individual cells and at the level of embryos directly indicates that all the embryos studied turned out to be "mosaics". This means that after editing in different cells of the same embryo, the genotype is different. The mosaic of embryos is currently one of the main and unresolved problems of editing people.
The same research group published a pioneering work two years ago on editing the genome of human embryos, which can be read here. In previous work, scientists tried to correct a mutation in the HBB gene using the "canonical" CRISPR/Cas9 system using homologous recombination to make a replacement in DNA. However, the efficiency of editing was very low – it was only in four cases for 54 embryos studied.
Many studies on human cells have since confirmed that using Cas9, the gene is easy to "break", but very difficult to "fix". The use of inactive Cas9 as a means of delivering other proteins has significantly expanded the capabilities of this system.
Portal "Eternal youth" http://vechnayamolodost.ru